Bone marrow-derived mesenchymal stem cells (Nanfang Hospital of Southern Medical University)

Bone marrow (BM) aspirates (40 mL) were diluted 1:1 with human MSC growth medium (low-glucose D-MEM with FBS (10%)). Mononuclear cells were separated using a 1077 mg/mL Ficoll-Paque solution and suspended in human MSC growth medium at a density of 5,000 cells/cm². Medium was replaced after three days and non-adherent cells were removed. Adherent cells were further cultured and medium was changed every three days. When reaching 70–80% confluency, cells were detached using a trypsin-EDTA solution and passaged at a ratio of 1:3. After passage 1, cells were plated at a mean density of 5×10³ cells/cm² in T175 cell culture flasks. Cells were harvested after 4–5 passages, washed three times in PBS-EDTA buffer and resuspended in 0.9% NaCl (w/v) for clinical use.